A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma
Abstract Background The level of antibodies against PfEMP1 is routinely quantified by the conventional microtitre enzyme-linked immunosorbent assay (ELISA). However, ELISA only measures one analyte at a time and requires a relatively large plasma volume if the complete antibody profile of the sample...
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ftdoajarticles:oai:doaj.org/article:c6bf9554a3b2417d88ba0c4419b3bdbc 2023-05-15T15:17:13+02:00 A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma Jensen Anja TR Lusingu John Theander Thor G Kurtis Jonathan Cham Gerald KK Turner Louise 2008-06-01T00:00:00Z https://doi.org/10.1186/1475-2875-7-108 https://doaj.org/article/c6bf9554a3b2417d88ba0c4419b3bdbc EN eng BMC http://www.malariajournal.com/content/7/1/108 https://doaj.org/toc/1475-2875 doi:10.1186/1475-2875-7-108 1475-2875 https://doaj.org/article/c6bf9554a3b2417d88ba0c4419b3bdbc Malaria Journal, Vol 7, Iss 1, p 108 (2008) Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 article 2008 ftdoajarticles https://doi.org/10.1186/1475-2875-7-108 2022-12-31T00:56:07Z Abstract Background The level of antibodies against PfEMP1 is routinely quantified by the conventional microtitre enzyme-linked immunosorbent assay (ELISA). However, ELISA only measures one analyte at a time and requires a relatively large plasma volume if the complete antibody profile of the sample is to be obtained. Furthermore, assay-to-assay variation and the problem of storage of antigen can influence ELISA results. The bead-based assay described here uses the BioPlex 100 (BioRad, Hercules, CA, USA) system which can quantify multiple antibodies simultaneously in a small plasma volume. Methods A total of twenty nine PfEMP1 domains were PCR amplified from 3D7 genomic DNA, expressed in the Baculovirus system and purified by metal-affinity chromatography. The antibody reactivity level to the recombinant PfEMP1 proteins in human hyper-immune plasma was measured by ELISA. In parallel, these recombinant PfEMP1 proteins were covalently coupled onto beads each having its own unique detection signal and the human hyper-immune plasma reactivity was detected for each individual protein using a BioPlex 100 system. Protein-coupled beads were analysed at two time points seven months apart, before and after lyophilization and the results compared to determine the effect of storage and lyophilization respectively on the beads. Multiplexed protein-coupled beads from twenty eight unique bead populations were evaluated on the BioPlex 100 system against pooled human hyper-immune plasma before and after lyophilization. Results The bead - based assay was sensitive, accurate and reproducible. Four recombinant PfEMP1 proteins C17, D5, D9 and D12, selected on the basis that they showed a spread of median fluorescent intensity (MFI) values from low to high when analysed by the bead-based assay were analysed by ELISA and the results from both analyses were highly correlated. The Spearman's rank correlation coefficients (Rho) were ≥ 0.86, (P < 0.0001) for all comparisons. Bead-based assays gave similar results regardless of whether ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic Rho ENVELOPE(-63.000,-63.000,-64.300,-64.300) Hercules ENVELOPE(161.450,161.450,-77.483,-77.483) Malaria Journal 7 1 |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
spellingShingle |
Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 Jensen Anja TR Lusingu John Theander Thor G Kurtis Jonathan Cham Gerald KK Turner Louise A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
description |
Abstract Background The level of antibodies against PfEMP1 is routinely quantified by the conventional microtitre enzyme-linked immunosorbent assay (ELISA). However, ELISA only measures one analyte at a time and requires a relatively large plasma volume if the complete antibody profile of the sample is to be obtained. Furthermore, assay-to-assay variation and the problem of storage of antigen can influence ELISA results. The bead-based assay described here uses the BioPlex 100 (BioRad, Hercules, CA, USA) system which can quantify multiple antibodies simultaneously in a small plasma volume. Methods A total of twenty nine PfEMP1 domains were PCR amplified from 3D7 genomic DNA, expressed in the Baculovirus system and purified by metal-affinity chromatography. The antibody reactivity level to the recombinant PfEMP1 proteins in human hyper-immune plasma was measured by ELISA. In parallel, these recombinant PfEMP1 proteins were covalently coupled onto beads each having its own unique detection signal and the human hyper-immune plasma reactivity was detected for each individual protein using a BioPlex 100 system. Protein-coupled beads were analysed at two time points seven months apart, before and after lyophilization and the results compared to determine the effect of storage and lyophilization respectively on the beads. Multiplexed protein-coupled beads from twenty eight unique bead populations were evaluated on the BioPlex 100 system against pooled human hyper-immune plasma before and after lyophilization. Results The bead - based assay was sensitive, accurate and reproducible. Four recombinant PfEMP1 proteins C17, D5, D9 and D12, selected on the basis that they showed a spread of median fluorescent intensity (MFI) values from low to high when analysed by the bead-based assay were analysed by ELISA and the results from both analyses were highly correlated. The Spearman's rank correlation coefficients (Rho) were ≥ 0.86, (P < 0.0001) for all comparisons. Bead-based assays gave similar results regardless of whether ... |
format |
Article in Journal/Newspaper |
author |
Jensen Anja TR Lusingu John Theander Thor G Kurtis Jonathan Cham Gerald KK Turner Louise |
author_facet |
Jensen Anja TR Lusingu John Theander Thor G Kurtis Jonathan Cham Gerald KK Turner Louise |
author_sort |
Jensen Anja TR |
title |
A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma |
title_short |
A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma |
title_full |
A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma |
title_fullStr |
A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma |
title_full_unstemmed |
A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma |
title_sort |
semi-automated multiplex high-throughput assay for measuring igg antibodies against plasmodium falciparum erythrocyte membrane protein 1 (pfemp1) domains in small volumes of plasma |
publisher |
BMC |
publishDate |
2008 |
url |
https://doi.org/10.1186/1475-2875-7-108 https://doaj.org/article/c6bf9554a3b2417d88ba0c4419b3bdbc |
long_lat |
ENVELOPE(-63.000,-63.000,-64.300,-64.300) ENVELOPE(161.450,161.450,-77.483,-77.483) |
geographic |
Arctic Rho Hercules |
geographic_facet |
Arctic Rho Hercules |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
Malaria Journal, Vol 7, Iss 1, p 108 (2008) |
op_relation |
http://www.malariajournal.com/content/7/1/108 https://doaj.org/toc/1475-2875 doi:10.1186/1475-2875-7-108 1475-2875 https://doaj.org/article/c6bf9554a3b2417d88ba0c4419b3bdbc |
op_doi |
https://doi.org/10.1186/1475-2875-7-108 |
container_title |
Malaria Journal |
container_volume |
7 |
container_issue |
1 |
_version_ |
1766347482694942720 |